Most cancer cells rely on altered metabolism to sustain rapid proliferation. The metabolic switch that occurs in cancer cells has been linked to specific oncogenes and tumor suppressors (myc, p53). Normal cells expressing p53 have high GLS2 (phosphate-activated glutaminase) activity that shunts glutamate through the TCA cycle and provides for glutathione (GSH) synthesis and antioxidant defense. These data suggest that regulation of glutamate flux contributes to the tumor suppressive actions of p53, and raise the possibility that dietary agents that mimic the effects of p53 on metabolism might be effective in cancer prevention. Through genomic profiling, we identified SLC1A1 (a glutamate transporter) and GLUL (glutamine synthetase) as vitamin D regulated genes suggesting that, like p53, vitamin D might act to reduce risk of transformation by regulation of glutamate metabolism. Hypothesis and Objectives: We hypothesize that regulation of SLC1A1 and GLUL by vitamin D alters glutamate/glutamine pools and suppresses the metabolic switch associated with tumorigenesis. This hypothesis predicts that vitamin D maintains high intracellular glutamate to enhance mitochondrial respiration and the TCA cycle and promote GSH synthesis. In Aim 1, the effects of physiological concentrations of 25- hydroxyvitamin D (25D) on metabolic gene expression and glutamate/glutamine metabolism in non- transformed mammary cells will be assessed in relation to GSH synthesis and proliferation. We will also conduct metabolomics analysis to identify vitamin D responsive pathways. In Aim 2, we will develop cellular models that mimic cancer-associated metabolic disturbances to test whether 25D can prevent changes in metabolism. In Aim 3, we will use animal models to determine whether dietary vitamin D alters SLC1A1 and GLUL expression/activity in normal mammary gland or in at-risk mammary tissue in relation to tumor prevention. Relevance: While some epidemiologic studies support a role for vitamin D in breast cancer prevention, questions remain regarding specific targets and mechanisms, tissue specificity, and optimal intakes of vitamin D to maximize potential benefits on cancer risk. The Institute of Medicine recently evaluated published literature on vitamin D and cancer and determined that additional research is needed to establish guidelines for vitamin D intake with respect to cancer prevention. In the proposed project, we will examine the relationship between vitamin D status and metabolism in mouse models of breast cancer using dietary manipulation to induce a wide range of serum 25D levels representative of human vitamin D deficiency, sufficiency and excess. Although our dietary studies will be conducted in mice, the conserved relationship between dietary vitamin D and serum 25D will provide a starting point for extrapolation to human populations.